Eukaryotic Translation Initiation Factor 4AIII (eIF4AIII) Is Functionally Distinct from eIF4AI and eIF4AII

Eukaryotic initiation factor 4A (eIF4A) is an RNA-dependent ATPase and ATP-dependent RNA helicase that is thought to melt the 5' proximal secondary structure of eukaryotic mRNAs to facilitate attachment of the 40S ribosomal subunit. eIF4A functions in a complex termed eIF4F with two other initiation factors (eIF4E and eIF4G). Two isoforms of eIF4A, eIF4AI and eIF4AII, which are encoded by two different genes, are functionally indistinguishable. A third member of the eIF4A family, eIF4AIII, whose human homolog exhibits 65% amino acid identity to human eIF4AI, has also been cloned from Xenopus and tobacco, but its function in translation has not been characterized. In this study, human eIF4AIII was characterized biochemically. While eIF4AIII, like eIF4AI, exhibits RNA-dependent ATPase activity and ATP-dependent RNA helicase activity, it fails to substitute for eIF4AI in an in vitro-reconstituted 40S ribosome binding assay. Instead, eIF4AIII inhibits translation in a reticulocyte lysate system. In addition, whereas eIF4AI binds independently to the middle and carboxy-terminal fragments of eIF4G, eIF4AIII binds to the middle fragment only. These functional differences between eIF4AI and eIF4AIII suggest that eIF4AIII might play an inhibitory role in translation under physiological conditions.     

Vaccinia Virus Protein Synthesis Has a Low Requirement for the Intact Translation Initiation Factor eIF4F, the Cap-Binding Complex, within Infected Cells

The role of the cap-binding complex, eIF4F, in the translation of vaccinia virus mRNAs has been analyzed within infected cells. Plasmid DNAs, which express dicistronic mRNAs containing a picornavirus internal ribosome entry site, produced within vaccinia virus-infected cells both β-glucuronidase and a cell surface-targeted single-chain antibody (sFv). Cells expressing sFv were selected from nonexpressing cells, enabling analysis of protein synthesis specifically within the transfected cells. Coexpression of poliovirus 2A or foot-and-mouth disease virus Lb proteases, which cleaved translation initiation factor eIF4G, greatly inhibited cap-dependent protein (β-glucuronidase) synthesis. Under these conditions, internal ribosome entry site-directed expression of sFv continued and cell selection was maintained. Furthermore, vaccinia virus protein synthesis persisted in the selected cells containing cleaved eIF4G. Thus, late vaccinia virus protein synthesis has a low requirement for the intact cap-binding complex eIF4F. This may be attributed to the short unstructured 5′ noncoding regions of the vaccinia virus mRNAs, possibly aided by the presence of poly(A) at both 5′ and 3′ termini.     

Identification of a Translation Initiation Factor 3 (eIF3) Core Complex, Conserved in Yeast and Mammals, That Interacts with eIF5

Only five of the nine subunits of human eukaryotic translation initiation factor 3 (eIF3) have recognizable homologs encoded in the Saccharomyces cerevisiae genome, and only two of these (Prt1p and Tif34p) were identified previously as subunits of yeast eIF3. We purified a polyhistidine-tagged form of Prt1p (His-Prt1p) by Ni²⁺ affinity and gel filtration chromatography and obtained a complex of ≈600 kDa composed of six polypeptides whose copurification was completely dependent on the polyhistidine tag on His-Prt1p. All five polypeptides associated with His-Prt1p were identified by mass spectrometry, and four were found to be the other putative homologs of human eIF3 subunits encoded in S. cerevisiae: YBR079c/Tif32p, Nip1p, Tif34p, and YDR429c/Tif35p. The fifth Prt1p-associated protein was eIF5, an initiation factor not previously known to interact with eIF3. The purified complex could rescue Met-tRNA_i^Met binding to 40S ribosomes in defective extracts from a prt1 mutant or extracts from which Nip1p had been depleted, indicating that it possesses a known biochemical activity of eIF3. These findings suggest that Tif32p, Nip1p, Prt1p, Tif34p, and Tif35p comprise an eIF3 core complex, conserved between yeast and mammals, that stably interacts with eIF5. Nip1p bound to eIF5 in yeast two-hybrid and in vitro protein binding assays. Interestingly, Sui1p also interacts with Nip1p, and both eIF5 and Sui1p have been implicated in accurate recognition of the AUG start codon. Thus, eIF5 and Sui1p may be recruited to the 40S ribosomes through physical interactions with the Nip1p subunit of eIF3.     

Translation Directed by Hepatitis A Virus IRES in the Absence of Active eIF4F Complex and eIF2

Translation directed by several picornavirus IRES elements can usually take place after cleavage of eIF4G by picornavirus proteases 2A^pro or L^pro. The hepatitis A virus (HAV) IRES is thought to be an exception to this rule because it requires intact eIF4F complex for translation. In line with previous results we report that poliovirus (PV) 2A^pro strongly blocks protein synthesis directed by HAV IRES. However, in contrast to previous findings we now demonstrate that eIF4G cleavage by foot-and-mouth disease virus (FMDV) L^pro strongly stimulates HAV IRES-driven translation. Thus, this is the first observation that 2A^pro and L^pro exhibit opposite effects to what was previously thought to be the case in HAV IRES. This effect has been observed both in hamster BHK and human hepatoma Huh7 cells. In addition, this stimulation of translation is also observed in cell free systems after addition of purified L^pro. Notably, in presence of this FMDV protease, translation directed by HAV IRES takes place when eIF2α has been inactivated by phosphorylation. Our present findings clearly demonstrate that protein synthesis directed by HAV IRES can occur when eIF4G has been cleaved and after inactivation of eIF2. Therefore, translation directed by HAV IRES without intact eIF4G and active eIF2 is similar to that observed with other picornavirus IRESs.     

Modulation of the helicase activity of eIF4A by eIF4B, eIF4H, and eIF4F.

Eukaryotic initiation factor (eIF) 4A is a DEAD box RNA helicase that works in conjunction with eIF4B, eIF4H, or as a subunit of eIF4F to unwind secondary structure in the 5'-untranslated region of mRNA, which facilitates binding of the mRNA to the 40 S ribosomal subunit. This study demonstrates how the helicase activity of eIF4A is modulated by eIF4B, eIF4H, or as a subunit of eIF4F. Results indicate that a linear relationship exists between the initial rate or amplitude of unwinding and duplex stability for all factor combinations tested. eIF4F, like eIF4A, behaves as a non-processive helicase. Either eIF4B or eIF4H stimulated the initial rate and amplitude of eIF4A-dependent duplex unwinding, and the magnitude of stimulation is dependent on duplex stability. Furthermore, eIF4A (or eIF4F) becomes a slightly processive helicase in the presence of eIF4B or eIF4H. All combinations of factors tested indicate that the rate of duplex unwinding is equivalent in the 5' --> 3' and 3' --> 5' directions. However, the optimal rate of unwinding was dependent on the length of the single-stranded region of the substrate when different combinations of factors were used. The combinations of eIF4A, eIF4A + eIF4B, eIF4A + eIF4H, and eIF4F showed differences in their ability to unwind chemically modified duplexes. A simple model of how eIF4B or eIF4H affects the duplex unwinding mechanism of eIF4A is proposed.     

Translation of mRNAs from Vesicular Stomatitis Virus and Vaccinia Virus Is Differentially Blocked in Cells with Depletion of eIF4GI and/or eIF4GII

Cytolytic viruses abrogate host protein synthesis to maximize the translation of their own mRNAs. In this study, we analyzed the eukaryotic initiation factor (eIF) 4G requirement for translation of vesicular stomatitis virus (VSV) and vaccinia virus (VV) mRNAs in HeLa cells using two different strategies: eIF4G depletion by small interfering RNAs or cleavage of eIF4G by expression of poliovirus 2A protease. Depletion of eIF4GI or eIF4GII moderately inhibits cellular protein synthesis, whereas silencing of both factors has only a slightly higher effect. Under these conditions, the extent of VSV protein synthesis is similar to that of nondepleted control cells, whereas VV expression is substantially reduced. Similar results were obtained when eIF4E was depleted. On the other hand, eIF4G cleavage by poliovirus 2A protease strongly inhibits translation of VV protein expression, whereas translation directed by VSV mRNAs is not abrogated, even though VSV mRNAs are capped. Therefore, the requirement for eIF4F activity is different for VV and VSV, suggesting that the molecular mechanism by which their mRNAs initiate their translation is also different. Consistent with these findings, eIF4GI does not colocalize with ribosomes in VSV-infected cells, while eIF2α locates at perinuclear sites coincident with ribosomes.     

A novel interaction of Cap-binding protein complexes eukaryotic initiation factor (eIF) 4F and eIF(iso)4F with a region in the 3'-untranslated region of satellite tobacco necrosis virus

Satellite tobacco necrosis virus (STNV) RNA is naturally uncapped at its 5' end and lacks polyadenylation at its 3' end. Despite lacking these two hallmarks of eukaryotic mRNAs, STNV-1 RNA is translated very efficiently. A approximately 130-nucleotide translational enhancer (TED), located 3' to the termination codon, is necessary for efficient cap-independent translation of STNV-1 RNA. The STNV-1 TED RNA fragment binds to the eukaryotic cap-binding complexes, initiation factor (eIF) 4F and eIF(iso)4F, as measured by nitrocellulose binding and fluorescence titration. STNV-1 TED is a potent inhibitor of in vitro translation when added in trans. This inhibition is reversed by the addition of eIF4F or eIF(iso)4F, and the subunits of eIF4F and eIF(iso)4F cross-link to STNV-1 TED, providing additional evidence that these factors interact directly with STNV-1 TED. Deletion mutagenesis of the STNV-1 TED indicates that a minimal region of approximately 100 nucleotides is necessary to promote cap-independent translation primarily through interaction with the cap binding subunits (eIF4E or eIF(iso)4E) of eIF4F or eIF(iso)4F.     

Initiation of mRNA Translation in Oncogenesis: The Role of eIF4E

The eukaryotic initiation factor 4E (eIF4E) is a key regulator of protein translation whose function is activated by the Akt and Ras proto-oncogenic signal transduction pathways. eIF4E enhances the translation of mRNAs encoding several genes involved in tumorigenesis and acts as a proto-oncogene, in vitro, when overexpressed in immortalized cells. Importantly, eIF4E is frequently found overexpressed in human cancers of multiple histological origins. However, in vivo evidence of the eIF4E neoplastic potential was lacking until now. Here we discuss recent findings that demonstrate eIF4E’s oncogenic role in vivo through direct genetic approaches in the mouse, and identify novel oncogenic functions for this initiation factor in cooperative tumorigenesis and response to therapy.     

Human Eukaryotic Initiation Factor 4G (eIF4G) Protein Binds to eIF3c, -d,and -e to Promote mRNA Recruitment to the Ribosome

Recruitment of mRNA to the 40S ribosomal subunit requires the coordinated interaction of a large number of translation initiation factors. In mammals, the direct interaction between eukaryotic initiation factor 4G (eIF4G) and eIF3 is thought to act as the molecular bridge between the mRNA cap-binding complex and the 40S subunit. A discrete ∼90 amino acid domain in eIF4G is responsible for binding to eIF3, but the identity of the eIF3 subunit(s) involved is less clear. The eIF3e subunit has been shown to directly bind eIF4G, but the potential role of other eIF3 subunits in stabilizing this interaction has not been investigated. It is also not clear if the eIF4A helicase plays a role in stabilizing the interaction between eIF4G and eIF3. Here, we have used a fluorescence anisotropy assay to demonstrate that eIF4G binds to eIF3 independently of eIF4A binding to the middle region of eIF4G. By using a site-specific cross-linking approach, we unexpectedly show that the eIF4G-binding surface in eIF3 is comprised of the -c, -d and -e subunits. Screening multiple cross-linker positions reveals that eIF4G contains two distinct eIF3-binding subdomains within the previously identified eIF3-binding domain. Finally, by employing an eIF4G-dependent translation assay, we establish that both of these subdomains are required for efficient mRNA recruitment to the ribosome and stimulate translation. Our study reveals unexpected complexity to the eIF3-eIF4G interaction that provides new insight into the regulation of mRNA recruitment to the human ribosome.     

Requirement of RNA Binding of Mammalian Eukaryotic Translation Initiation Factor 4GI (eIF4GI) for Efficient Interaction of eIF4E with the mRNA Cap

Eukaryotic mRNAs possess a 5′-terminal cap structure (cap), m⁷GpppN, which facilitates ribosome binding. The cap is bound by eukaryotic translation initiation factor 4F (eIF4F), which is composed of eIF4E, eIF4G, and eIF4A. eIF4E is the cap-binding subunit, eIF4A is an RNA helicase, and eIF4G is a scaffolding protein that bridges between the mRNA and ribosome. eIF4G contains an RNA-binding domain, which was suggested to stimulate eIF4E interaction with the cap in mammals. In Saccharomyces cerevisiae, however, such an effect was not observed. Here, we used recombinant proteins to reconstitute the cap binding of the mammalian eIF4E-eIF4GI complex to investigate the importance of the RNA-binding region of eIF4GI for cap interaction with eIF4E. We demonstrate that chemical cross-linking of eIF4E to the cap structure is dramatically enhanced by eIF4GI fragments possessing RNA-binding activity. Furthermore, the fusion of RNA recognition motif 1 (RRM1) of the La autoantigen to the N terminus of eIF4GI confers enhanced association between the cap structure and eIF4E. These results demonstrate that eIF4GI serves to anchor eIF4E to the mRNA and enhance its interaction with the cap structure.The cap structure, m⁷GpppN, is present at the 5′ terminus of all nuclear transcribed eukaryotic mRNAs. Cap-dependent binding of the ribosome to mRNA is mediated by the cap-binding protein eukaryotic translation initiation factor 4E (eIF4E), which forms a complex termed eIF4F together with eIF4G and eIF4A. Mammalian eIF4G, which has two isoforms, eIF4GI and eIF4GII, is a modular, multifunctional protein that binds to poly(A)-binding protein (PABP) (14) and eIF4E (18, 20) via the N-terminal third region. Mammalian eIF4G binds to eIF4A and eIF3 (15) via the middle third region and to eIF4A and Mnk protein kinase at the C-terminal region. eIF4GI also possesses an RNA-binding sequence (2, 9, 33) in the middle region. There are two RNA-binding sites on eIF4GI; one is located amino terminal to the first HEAT domain, and the other is located within the first HEAT domain (23). Mammalian and Saccharomyces cerevisiae eIF4E are similar in size (24 kDa), but mammalian eIF4GI (220 kDa) is larger than its yeast counterpart (150 kDa), as the latter lacks a C-terminal domain corresponding to mammalian eIF4GI (38).The affinity of eIF4E for the cap structure has been a matter of dispute for some time. The earlier works of Carberry et al. (4) and Ueda et al. (39) estimated the equilibrium dissociation constant (K_d) of the eIF4E-cap complex by fluorescence titration to be 2 × 10⁻⁶ to 5 × 10⁻⁶ M depending on the nature of the cap analog. Later on, development of a new methodology for the fluorescence titration experiments yielded K_d values of 10⁻⁷ to 10⁻⁸ (29, 41). The source of the difference with the previous reports was thoroughly analyzed (29, 30). The interaction between the cap structure and eIF4E is dramatically enhanced by eIF4GI. This was first reported by showing that cross-linking of mammalian eIF4E to the cap structure is more efficient when it is a subunit of the eIF4F complex (19) or when it is complexed to eIF4GI (11). A similar enhancement of the binding of eIF4E to the cap structure was observed in yeast (40). However, two very different mechanisms were proposed to explain these observations. For the mammalian system, it was postulated that the middle segment of eIF4GI, which binds RNA, stabilizes the eIF4E interaction with the cap structure (11). This model was based primarily on the finding that in poliovirus-infected cells, eIF4GI is cleaved between its N-terminal third and the middle third, and consequently, eIF4E remains attached to the N-terminal eIF4GI fragment lacking the RNA-binding region. Under these conditions, cross-linking of eIF4E to the cap structure was poor (19, 31). In contrast, in yeast, a strong interaction between the cap structure and eIF4E was achieved using an eIF4G fragment containing the eIF4E-binding site that lacks the RNA-binding region (34, 40). Also, the yeast eIF4G fragment from amino acids 393 to 490 (fragment 393-490), which does not contain the RNA-binding site, forms a right-handed helical ring that wraps around the N terminus of eIF4E. This conformational change was suggested in turn to engender an allosteric enhancement of the association of eIF4E with the cap structure (10). Such an interaction between mammalian eIF4GI and eIF4E has not been reported.To understand the mechanism by which eIF4GI stimulates the interaction of eIF4E with the cap structure in mammals, we reconstituted the eIF4E-cap recognition activity in vitro with purified eIF4E and eIF4GI recombinant proteins. Using a chemical cross-linking assay, we demonstrate that only mammalian eIF4GI fragments possessing RNA-binding activity enhance the cross-linking of eIF4E to the cap structure. Our data provide new insight into the mechanism of cap recognition by the eIF4E-eIF4GI complex.     

Human Eukaryotic Initiation Factor 2 (eIF2)-GTP-Met-tRNAi Ternary Complex and eIF3 Stabilize the 43 S Preinitiation Complex

The formation of a stable 43 S preinitiation complex (PIC) must occur to enable successful mRNA recruitment. However, the contributions of eIF1, eIF1A, eIF3, and the eIF2-GTP-Met-tRNA_i ternary complex (TC) in stabilizing the 43 S PIC are poorly defined. We have reconstituted the human 43 S PIC and used fluorescence anisotropy to systematically measure the affinity of eIF1, eIF1A, and eIF3j in the presence of different combinations of 43 S PIC components. Our data reveal a complicated network of interactions that result in high affinity binding of all 43 S PIC components with the 40 S subunit. Human eIF1 and eIF1A bind cooperatively to the 40 S subunit, revealing an evolutionarily conserved interaction. Negative cooperativity is observed between the binding of eIF3j and the binding of eIF1, eIF1A, and TC with the 40 S subunit. To overcome this, eIF3 dramatically increases the affinity of eIF1 and eIF3j for the 40 S subunit. Recruitment of TC also increases the affinity of eIF1 for the 40 S subunit, but this interaction has an important indirect role in increasing the affinity of eIF1A for the 40 S subunit. Together, our data provide a more complete thermodynamic framework of the human 43 S PIC and reveal important interactions between its components to maintain its stability.     

Mitotic Phosphorylation of Eukaryotic Initiation Factor 4G1 (eIF4G1) at Ser1232 by Cdk1:Cyclin B Inhibits eIF4A Helicase Complex Binding with RNA

During mitosis, global translation is suppressed, while synthesis of proteins with vital mitotic roles must go on. Prior evidence suggests that the mitotic translation shift involves control of initiation. Yet, no signals specifically targeting translation initiation factors during mitosis have been identified. We used phosphoproteomics to investigate the central translation initiation scaffold and “ribosome adaptor,” eukaryotic initiation factor 4G1 (eIF4G1) in interphase or nocodazole-arrested mitotic cells. This approach and kinase inhibition assays, in vitro phosphorylation with recombinant kinase, and kinase depletion-reconstitution experiments revealed that Ser1232 in eIF4G1 is phosphorylated by cyclin-dependent kinase 1 (Cdk1):cyclin B during mitosis. Ser1232 is located in an unstructured region of the C-terminal portion of eIF4G1 that coordinates assembly of the eIF4G/-4A/-4B helicase complex and binding of the mitogen-activated protein kinase (MAPK) signal-integrating kinase, Mnk. Intense phosphorylation of Ser1232 in mitosis strongly enhanced the interactions of eIF4A with HEAT domain 2 of eIF4G and decreased association of eIF4G/-4A with RNA. Our findings implicate phosphorylation of eIF4G1(Ser1232) by Cdk1:cyclin B and its inhibitory effects on eIF4A helicase activity in the mitotic translation initiation shift.     

The Eukaryotic Initiation Factor (eIF) 4G HEAT Domain Promotes Translation Re-initiation in Yeast Both Dependent on and Independent of eIF4A mRNA Helicase

Translation re-initiation provides the molecular basis for translational control of mammalian ATF4 and yeast GCN4 mediated by short upstream open reading (uORFs) in response to eIF2 phosphorylation. eIF4G is the major adaptor subunit of eIF4F that binds the cap-binding subunit eIF4E and the mRNA helicase eIF4A and is also required for re-initiation in mammals. Here we show that the yeast eIF4G2 mutations altering eIF4E- and eIF4A-binding sites increase re-initiation at GCN4 and impair recognition of the start codons of uORF1 or uORF4 located after uORF1. The increase in re-initiation at GCN4 was partially suppressed by increasing the distance between uORF1 and GCN4, suggesting that the mutations decrease the migration rate of the scanning ribosome in the GCN4 leader. Interestingly, eIF4E overexpression suppressed both the phenotypes caused by the mutation altering eIF4E-binding site. Thus, eIF4F is required for accurate AUG selection and re-initiation also in yeast, and the eIF4G interaction with the mRNA-cap appears to promote eIF4F re-acquisition by the re-initiating 40 S subunit. However, eIF4A overexpression suppressed the impaired AUG recognition but not the increase in re-initiation caused by the mutations altering eIF4A-binding site. These results not only provide evidence that mRNA unwinding by eIF4A stimulates start codon recognition, but also suggest that the eIF4A-binding site on eIF4G made of the HEAT domain stimulates the ribosomal scanning independent of eIF4A. Based on the RNA-binding activities identified within the unstructured segments flanking the eIF4G2 HEAT domain, we discuss the role of the HEAT domain in scanning beyond loading eIF4A onto the pre-initiation complex.     

The phosphorylation state of poly(A)-binding protein specifies its binding to poly(A) RNA and its interaction with eukaryotic initiation factor (eIF) 4F, eIFiso4F, and eIF4B

The poly(A)-binding protein (PABP) interacts with the eukaryotic initiation factor (eIF) 4G (or eIFiso4G), the large subunit of eIF4F (or eIFiso4F) to promote translation initiation. In plants, PABP also interacts with eIF4B, a factor that assists eIF4F function. PABP is a phosphoprotein, although the function of its phosphorylation has not been previously investigated. In this study, we have purified the phosphorylated and hypophosphorylated isoforms of PABP from wheat to examine whether its phosphorylation state affects its binding to poly(A) RNA and its interaction with eIF4G, eIFiso4G, or eIF4B. Phosphorylated PABP exhibited cooperative binding to poly(A) RNA even under non-stoichiometric binding conditions, whereas multiple molecules of hypophosphorylated PABP bound to poly(A) RNA only after free poly(A) RNA was no longer available. Together, phosphorylated and hypophosphorylated PABP exhibited synergistic binding. eIF4B interacted with PABP in a phosphorylation state-specific manner; native eIF4B increased the RNA binding activity specifically of phosphorylated PABP and was greater than 14-fold more effective than was recombinant eIF4B, whereas eIF4F promoted the cooperative binding of hypophosphorylated PABP. These data suggest that the phosphorylation state of PABP specifies the type of binding to poly(A) RNA and its interaction with its partner proteins.     

20S proteasome differentially alters translation of different mRNAs via the cleavage of eIF4F and eIF3

The molecular basis for coordinated regulation of protein synthesis and degradation is not understood. Here we report that the 20S proteasome endoproteolytically cleaves the translation initiation factors eIF4G, a subunit of eIF4F, and eIF3a, a subunit of eIF3. The cleavage of eIF4G or eIF3a differentially affects the assembly of ribosomal preinitiation complexes on different cellular and viral mRNAs in an in vitro system containing pure components. Inhibition of proteolytic activity of the 20S proteasome with specific inhibitors prevents cleavage of both factors in vitro and in vivo, restores assembly of ribosomal complexes in vitro, and differentially affects translation of different mRNAs in vivo. These studies demonstrate the importance of the endoproteolytic activity of proteasomes in regulation of cellular processes and suggest a link between protein synthesis and degradation.     

Selective modification of eukaryotic initiation factor 4F (eIF4F) at the onset of cell differentiation: recruitment of eIF4GII and long-lasting phosphorylation of eIF4E

mRNA translation is mainly regulated at the level of initiation, a process that involves the synergistic action of the 5' cap structure and the 3' poly(A) tail at the ends of eukaryotic mRNA. The eukaryote initiation factor 4G(eIF4G) is a pivotal scaffold protein that forms a critical link between mRNA cap structure, poly(A) tail, and the small ribosomal subunit. There are two functional homologs of eIF4G in mammals, the original eIF4G, renamed eIF4GI, and eIF4GII that functionally complements eIF4GI. To date, biochemical and functional analysis have not identified differential activities for eIF4GI and eIF4GII. In this report, we demonstrate that eIF4GII, but not eIF4GI, is selectively recruited to capped mRNA at the onset of cell differentiation. This recruitment is coincident with a strong and long-lasting phosphorylation of eIF4E and the release of 4E-BP1, a suppressor of eIF4E function, from the cap structure, without a concomitant change in 4E-BP1's phosphorylation. Our data further indicate that cytokines such as thrombopoietin can differentially regulate eIF4GI/II activities. These results provide the first evidence that eIF4GI/II does fulfill selective roles in mammalian cells.     

Coordinated Movements of Eukaryotic Translation Initiation Factors eIF1, eIF1A,and eIF5 Trigger Phosphate Release from eIF2 in Response to Start Codon Recognition by the Ribosomal Preinitiation Complex

Accurate recognition of the start codon in an mRNA by the eukaryotic translation preinitiation complex (PIC) is essential for proper gene expression. The process is mediated by eukaryotic translation initiation factors (eIFs) in conjunction with the 40 S ribosomal subunit and (initiator) tRNA_i. Here, we provide evidence that the C-terminal tail (CTT) of eIF1A, which we previously implicated in start codon recognition, moves closer to the N-terminal domain of eIF5 when the PIC encounters an AUG codon. Importantly, this movement is coupled to dissociation of eIF1 from the PIC, a critical event in start codon recognition, and is dependent on the scanning enhancer elements in the eIF1A CTT. The data further indicate that eIF1 dissociation must be accompanied by the movement of the eIF1A CTT toward eIF5 in order to trigger release of phosphate from eIF2, which converts the latter to its GDP-bound state. Our results also suggest that release of eIF1 from the PIC and movement of the CTT of eIF1A are triggered by the same event, most likely accommodation of tRNA_i in the P site of the 40 S subunit driven by base pairing between the start codon in the mRNA and the anticodon in tRNA_i. Finally, we show that the C-terminal domain of eIF5 is responsible for the factor''s activity in antagonizing eIF1 binding to the PIC. Together, our data provide a more complete picture of the chain of molecular events that is triggered when the scanning PIC encounters an AUG start codon in the mRNA.     

Wheat germ poly(A)-binding protein increases the ATPase and the RNA helicase activity of translation initiation factors eIF4A, eIF4B, and eIF-iso4F

Recent studies demonstrated that wheat germ poly(A)-binding protein (PABP) interacted with translation eukaryotic initiation factor (eIF)-iso4G and eIF4B, and these interactions increased the poly(A) binding activity of PABP (Le, H., Tanguay, R. L., Balasta, M. L., Wei, C. C., Browning, K. S., Metz, A. M., Goss, D. J., and Gallie, D. R. (1997) J. Biol. Chem. 272, 16247-16255) and the cap binding activity of eIF-iso4F (Wei, C. C., Balasta, M. L., Ren, J., and Goss, D. J. (1998) Biochemistry 37, 1910-1916). We report here that the interaction between PABP and eIF-iso4G has a substantial effect on the ATPase activity and RNA helicase activity of (eIF4A + eIF4B + eIF-iso4F) complex. ATPase kinetic assays show, in the presence of poly(U), PABP can increase the parameter (k(cat)/K(m)) by 3.5-fold with a 2-fold decrease of K(m) for the (eIF4A + eIF-iso4F) complex. In the presence of globin messenger RNA, the ATPase activity of the complex (eIF4A + eIF-iso4F) was increased 2-fold by the presence of PABP. RNA helicase assays demonstrated that the presence of PABP enhanced the RNA duplex unwinding activity of the initiation factor complex. These results suggest that, in terms of the scanning model of translation initiation, PABP may enhance the mRNA scanning rate of the complex formed by eIF4A, eIF4B, and eIF4F or eIF-(iso)4F and increase the rate of translation.